Human Granulocyte Colony Stimulating Factor (h-G-CSF) is a 20 kDa glycoprotein, produced naturally by stromal cells, macrophages, fibroblasts and monocytes. Its production increases upon exposition to endotoxins and during infection. G-CSF acts in the bone marrow, where it binds with high affinity to G-CSF receptors (G-CSFR), expressed on neutrophile precursor cells, by inducing their proliferation and differentiation into mature anti-infective neutrophiles, but without significant hemopoietic effects on other hematic cell lines.
The main native G-CSF isoform is a 174-aminoacid polypeptide having four cysteine residues employed in two disulfide bonds, a free cysteine residue at position 17 and a glycosilation site on the oxygen (O-linked glycosilation) of the threonine residue side chain at position 133. It has been reported that although glycosilation is not necessary to establish an efficient receptorial bond or for G-CSF biological activity (N. A. Nicola, “Hemopoietic Cell Growth Factors and their Receptors”, Ann. Rev. Biochem., 58, 45-77, 1989), the presence of the single O-glycosidic chain improves the physical and enzymatic stability of G-CSF (M. Oh-eda et al., “O-linked Sugar Chain of Human Granulocyte Stimulating Factor Protects it against Polymerization and Denaturation Allowing it to Retain its Biological Activity”, J. Biol. Chem., 265, 11432-11435, 1990; C. R. D. Carter et al., “Human Serum Inactivates Non-Glycosylated but not Glycosylated Granulocyte Colony Stimulating Factor by a Protease Dependent Mechanism: Significance of Carbohydrates on the Glycosylated Molecule”, Biologicals, 32, 37-47, 2004). Human G-CSF production by genetic engineering techniques has lead to the development of new therapies to treat several kinds of neutropenia, both primary and secondary. In particular, recombinant G-CSF compounds are prescribed in the hospital setting for the following therapies:                shortening of neutropenia and related infective and febrile phenomena, in high-risk patients being treated with myelotoxic antitumoural drugs or myeloablation followed by bone marrow transplant;        mobilization of peripheral blood progenitor cells to be employed in autologous cell transplant, in patients undergoing myelosuppression or myeloablation, possibly followed by bone marrow transplant;        treatment of patients suffering from congenital or idiopathic neutropenia, showing severe reduction of neutrophile plasmatic concentration, as well as infections and fever;        treatment of neutropenia and bacterial infections, which develop in late-stage HIV infection patients.        
The particular interest shown in treating patients affected by different types of neutropenia with G-CSF has stimulated the development of recombinant compounds, produced both in mammalian and bacterial cell systems. In fact, at least three variants of recombinant G-CSF are available in several countries for therapeutic use:                a glycosilated form, called lenograstim, expressed in mammalian cells and engineered as a 174-aminoacid polypeptide chain identical to the native protein polypeptide chain and including an O-linked oligosaccharide moiety on the threonine residue at position 133;        a non-glycosilated form, called filgrastim, expressed in bacterial cells as a 175-aminoacid polypeptide chain identical to the native protein except for an additional methionyl residue at the N-terminal (met-G-CSF);        a non-glycosilated form, called nartograstim, expressed in bacterial cells as a 175-aminoacid polypeptide chain (met-G-CSF) which differs from the native protein polypeptide chain by the additional methionyl residue at the N-terminal, as well as substitution for Thr 2, Leu 4, Gly 5, Pro 6 and Cys 18 residues with, respectively, Ala, Thr, Tyr, Arg and Ser residues.        
A limit, well-known in the clinical application of the various recombinant G-CSF derivatives, is the short circulating permanence in the bloodstream after parenteral administration, with a pharmacokinetic half-life (t1/2) of 3-4 hours. As a consequence, the dosage of recombinant G-CSF prescribed, for example, to reduce the development of infections in patients having non-myeloid tumours and undergoing myelosuppressive chemotherapy, consists of daily administration of subcutaneous injections of 5 microgram/kg/die G-CSF for the duration of the chemotherapy cycle, amounting to 10-14 injections per cycle.
The G-CSF pharmacokinetic profile, as with the majority of cytokines, is regulated by a non-specific and nonsaturable renal clearance mechanism (and, to a lesser degree, hepatic clearance), in addition to a specific and saturable mechanism of internalization and partial degradation mediated by cells expressing the G-CSF receptor.
Since renal clearance is related to the size of the protein molecule, one way of reducing renal ultrafiltration is to increase the molecular size and/or the hydrodynamic volume.
This is a very common problem in the field of therapeutic proteins and several solutions have been proposed, such as fusion of the therapeutic protein to carrier proteins (for example, immunoglobulin or albumin); incorporation of the active ingredient in slow-release polymer nano- and microspheres; and covalent bond protein conjugation to biocompatible, high molecular weight polymers.
In particular, in the area of covalent protein-polymer conjugation, the so-called PEGylation reaction has been extensively employed, where the chosen protein is covalently bound to one or more linear or branched poly(ethylene glycol) (PEG) chains, having a molecular weight ranging from 1,000-2,000 Da to 20,000-40,000 Da or even higher. In general, PEGylated proteins show lower renal clearance rates, as well as higher stability and reduced immunogenicity. When PEG is suitably bound to a polypeptide, its hydrodynamic volume and physico-chemical properties are modified, while fundamental biological functions, such as in vitro activity or receptor recognition, may remain unchanged, undergo a slight reduction or, in some cases, be completely suppressed. PEG conjugation masks the protein surface and increases its molecular size, thus decreasing renal ultrafiltration, preventing attachment of antibodies or antigen processing cells and reducing proteolytic enzyme degradation. Finally, PEG conjugation confers the physico-chemical properties of PEG and, therefore, peptide and non-peptide drug biodistribution and solubility are similarly modified. As an alternative to PEG for protein conjugation, other linear or branched biocompatible polymers, such as dextran, poly(vinylpyrrolidone), poly(acryloylmorpholine) or polysaccharides may be employed.
For a survey of commonly employed chemical PEGylation techniques and results, reference is made to the following:                S. Zalipsky, Chemistry of Polyethylene Glycol Conjugates with Biologically Active Molecules, Adv. Drug Deliv. Rev., 16, 157-182, 1995;        F. M. Veronese, Peptide and Protein PEGylation: a Review of Problems and Solutions, Biomaterials, 22, 405-417, 2001.        
G-CSF covalent conjugation to high molecular weight, biocompatible polymers has been described, for example, in several scientific articles and patents, some of which are briefly summarized below.
WO 89/06546 describes a G-CSF genetic variant, chemically conjugated to polymer chains of poly(ethylene glycol) or poly(propylene glycol) which maintains biological activity and shows an enhanced bloodstream half-life.
WO 90/06952 describes a G-CSF modification with PEG chains by chemically binding the amino and carboxyl groups of aminoacid side chains to yield a long half-life PEG-G-CSF conjugate.
WO 00/44785 describes G-CSF derivatives, chemically bound to 1-15 polymer PEG chains where stability, solubility and bloodstream circulation are improved after in vivo administration.
EP0335423 describes PEG-G-CSF chemical conjugates showing different structural, physico-chemical and biological properties.
While the abovementioned conjugates, mainly obtained by nonselective conjugation with G-CSF amino or carboxyl groups, are usually conjugated isoform mixtures, G-CSF chemical conjugates have been developed to yield essentially site-specific, mono-conjugate derivatives, as reported below.
U.S. Pat. No. 5,985,265 describes a method for polymer compound conjugation to the α-amino group of the N-terminal aminoacid residue of a polypeptide chain, which can be obtained both by an amide bond between the polymer and the protein, and, preferably, by an amine bond between the polymer and the protein through a reductive alkylation reaction (O. Kinstler et al., Mono-N-terminal Poly-(Ethylen Glycol)-Protein Conjugates, Adv. Drug Deliv. Rev., 54, 477-485, 2002). Application of this technology has, therefore, allowed development of a met-G-CSF conjugated to a 20 kDa linear PEG on the α-amino group of the N-terminal methionyl by a pH 5 reductive alkylation reaction with a monomethoxy-PEG chain functionalized with propionaldehyde; this product has been marketed with the international non-proprietary name of PEG-filgrastim and the registered brand name of Neulasta® (O. Kinstler et al., Characterization and Stability of N-terminally PEGylated rhG-CSF, Pharmac. Res., 13, 996-1002, 1996).
Another protein residue, which can potentially give rise to site-specific conjugates, is the cysteine thiol group, a highly reactive moiety to PEG molecules functionalized with residues forming a covalent bond with the thiol radical (M. J. Roberts et al., Chemistry for Peptide and Protein PEGylation, Adv. Drug Deliv. Rev., 54, 459-476, 2002). Since most proteins do not have a free cysteine residue (that is, not involved in a disulfide bond), it is possible to site-specifically conjugate polymer and protein by inserting into the polypeptide chain, through site-specific mutagenesis, a cysteine residue which will then permit reaction with the polymer functionalized with the cysteine thiol reactive group, as described for a series of G-CSF mutants (M. S. Rosendahl et al., Site-specific Protein PEGylation. Application to Cysteine Analogs of Recombinant Human Granulocyte Colony-Stimulating Factor, BioProcess Internat., 3 (4), 52-60, 2005).
According to a partially alternative approach, WO 2005/099769A2 describes r-h-G-CSF conjugation on the native cysteine thiol group not involved in disulfide bonds (Cys17), after partial protein denaturation, so that the free —SH moiety, otherwise masked in a hydrophobic pocket, is exposed to the solvent.
As well as the various abovementioned chemical conjugation techniques, enzymatic procedures have been described, to bind polymer and protein. These are based on the employment of transglutaminase enzymes, both prokaryotic and eukaryotic, to catalyze the transfer of a polymer, functionalized with a primary amino group, to the acyl groups of glutamine residues, naturally present in the polypeptide chain of interest or inserted via site-specific mutagenesis reactions (H. Sato, Enzymatic Procedure for Site-Specific PEGylation of Proteins, Adv. Drug Deliv. Rev., 54, 487-504, 2002).
Therefore, for instance, both EP785276 and U.S. Pat. No. 6,010,871 describe the use of a microbial transglutaminase (MTG) to insert polymer chains in peptides and proteins with at least one glutamine residue in their aminoacid sequence. In these patents, although examples are given of mono-substitution on some model proteins, it is not clear if the substitutions are also site-specific, meaning whether they yield a single molecular form or a positional isomer mixture where, though mono-substituted, the polymer chains are bound to different glutamines.